Cancer is one of the most deadly threats to human health. In the U.S. alone, cancer affects nearly 1.3 million new patients each year, and is the second leading cause of death after cardiovascular disease, accounting for approximately 1 in 4 deaths. Solid tumors are responsible for most of those deaths. Although there have been significant advances in the medical treatment of certain cancers, the overall 5-year survival rate for all cancers has improved only by about 10% in the past 20 years. Cancers, or malignant tumors, metastasize and grow rapidly in an uncontrolled manner, making treatment extremely difficult.
There are numerous unmet therapeutic needs in the treatment of solid and liquid tumors. ROR1, receptor tyrosine kinase-like orphan receptor 1, is an embryonic protein that is highly expressed in many cancer types, including CLL, carcinoma of the breast, glioblastoma, lung adenocarcinoma and sarcomas (Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, and fibrosarcoma), and is generally absent in normal tissues (Suping Zhang, et al., 2012, The OncoEmbryonic Antigen ROR1 Is Expressed by a Variety of Human Cancers. Am J Pathol, 181: 1903-1910, Ashwini Balakrishnan, et al., 2017, Analysis of ROR1 Protein Expression in Human Cancer and Normal Tissues., Clin Cancer Res 23:3061-3071, Borcherding, Nicholas et al., 2017, ROR1, an Embryonic Protein with an Emerging Role in Cancer Biology. Protein & Cell 5.7 (2014): 496-502). ROR1 has three splice variants, including a 104 kDa (up to 120 kDa depending on glycosylation) transmembrane glycoprotein comprised of 937 amino acids (1-29 signal peptide), and 2 smaller variants of intracellular and secreted forms (GeneBank NP_005003, Masiakowski, P., and Carroll, R. D., 1992, A Novel Family of Cell Surface Receptors with Tyrosine Kinase-like Domain, J Biol Chem 36: 26181-26190.). The presence of ROR1 on the surface of transformed cells indicates that targeting ROR1 will enable novel cancer treatments to be developed for a range of liquid cancer such as chronic lymphocytic leukemia (CLL) and other solid tumors (Borcherding, N., Kusner, D. et al., 2014, ROR1, an embryonic protein with an emerging role in cancer biology. Protein & Cell, 5:496-502).
Though generally absent in adult tissues, at least one report found ROR1 expression in parathyroid; pancreatic islets; and regions of the esophagus, stomach, and duodenum (Ashwini Balakrishnan, et al., 2017, Analysis of ROR1 Protein Expression in Human Cancer and Normal Tissues., Clin Cancer Res 23:3061-3071), warranting caution in clinical application of ROR1-targeted anti-cancer therapies. ROR1 receptor contains a cytosolic protein kinase domain, which, according to some reports, participates in Wnt and EGFR signaling (Borcherding, N., Kusner, D. et al., 2014, ROR1, an embryonic protein with an emerging role in cancer biology. Protein & Cell, 5:496-502). In tumors, ROR1 can induce epithelial to mesenchymal transition (EMT), and promote tumor proliferation, aggressiveness, and metastases formation, and mediate resistance to apoptosis (Yamaguchi, Tomoya, et al., 2012, “NKX2-1/TITF1/TTF-1-Induced ROR1 is required to sustain EGFR survival signaling in lung adenocarcinoma.” Cancer cell 21.3: 348-361; Borcherding, N., Kusner, D. et al., 2014, ROR1, an embryonic protein with an emerging role in cancer biology. Protein & Cell, 5:496-502). Its role in contributing to tumor phenotype indicates that it may serve an important function in tumor initiation or progression and therefore is a driver protein.
Earlier approaches in cancer treatment include surgery, radiation therapy, chemotherapy, and, for blood tumors—bone marrow transplant. However, the present first line treatments warrant further improvement. Such improvements are sought by the novel immunotherapeutic strategies. Ongoing pre-clinical investigations and clinical trials investigate targeting ROR1 antigen have been developed using multiple modalities. T lymphocytes expressing ROR1-specific CARs have been tested both in murine and non-human primate systems (Huang X, Park H, Greene J, Pao J, Mulvey E, Zhou S X, et al., 2015, IGF1R- and ROR1-Specific CAR T Cells as a Potential Therapy for High Risk Sarcomas. PLoS ONE 10(7): e0133152; Hudecek M, Schmitt T M, Baskar S, Lupo-Stanghellini M T, Nishida T, Yamamoto T N, Bleakley M, Turtle C J, Chang W C, Greisman H A, Wood B, Maloney D G, Jensen M C, Rader C, Riddell S R, 2010, The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor. Blood 116:4532-41.). The lack of toxicity in non-human primates lends confidence that human studies can be approached (Berger, C., et al., 2015, Safety of targeting ROR1 in primates with chimeric antigen receptor-modified T cells. Cancer Immunol Res 3: 2016-216.). Both unmodified, and immunotoxin-linked antibodies to ROR1 have also been proposed for therapeutic use (Yang, Jiahui, et al., 2011, “Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies.” PloS One 6.6: e21018; Baskar, Sivasubramanian, et al., 2012, “Targeting malignant B cells with an immunotoxin against ROR1.” MAbs, 4:3, 349-361.). The present standard of care for B-lineage leukemias may consists of remission induction treatment by high dose of chemotherapy or radiation, followed by consolidation, and may feature stem cell transplantation and additional courses of chemotherapy as needed (see the world wide web at cancer.gov). High toxicity associated with these treatments, as well as the risk of complications, such as relapse, secondary malignancy, or GVHD, motivate the search for better therapeutic alternatives. Current open clinical trials include ROR1-targeted T cells for hematologic malignancy (Genetically Modified T-Cell Therapy in Treating Patients with Advanced ROR1+ Malignancies, NCT02706392, Sponsor: Fred Hutchinson Cancer Research Center, ClinicalTrials.gov accessed Sep. 20, 2017.), and ROR1-specific antibody for breast cancer given in the context of chemotherapy (Study of Circumtuzumab and Paclitaxel for Metastatic or Locally Advanced, Unresectable Breast Cancer, NCT02776917, Sponsor: Barbara Parker, MD, University of California, San Diego, ClinicalTrials.gov accessed Sep. 20, 2017).
Chimeric Antigen Receptors (CARs) are hybrid molecules comprising three essential units: (1) an extracellular antigen-binding motif, (2) linking/transmembrane motifs, and (3) intracellular T-cell signaling motifs (Long A H, Haso W M, Orentas R J. Lessons learned from a highly-active CD22-specific chimeric antigen receptor, Oncolmmunology. 2013; 2 (4):e23621). The antigen-binding motif of a CAR is commonly fashioned after a single chain Fragment variable (ScFv), the minimal binding domain of an immunoglobulin (Ig) molecule. Alternate antigen-binding motifs, such as receptor ligands (i.e., IL-13 has been engineered to bind tumor expressed IL-13 receptor), intact immune receptors, library-derived peptides, and innate immune system effector molecules (such as NKG2D) also have been engineered. Alternate cell targets for CAR expression (such as NK or gamma-delta T cells) are also under development (Brown C E et al Clin Cancer Res. 2012; 18(8):2199-209; Lehner M et al. PLoS One. 2012; 7 (2):e31210). There remains significant work to be done with regard to defining the most active T-cell population to transduce with CAR vectors, determining the optimal culture and expansion techniques, and defining the molecular details of the CAR protein structure itself.
The linking motifs of a CAR can be a relatively stable structural domain, such as the constant domain of IgG, or designed to be an extended flexible linker. Structural motifs, such as those derived from IgG constant domains, can be used to extend the ScFv binding domain away from the T-cell plasma membrane surface. This may be important for some tumor targets where the binding domain is particularly close to the tumor cell surface membrane (such as for the disialoganglioside GD2; Orentas et al., unpublished observations). To date, the signaling motifs used in CARs always include the CD3-ζ chain because this core motif is the key signal for T cell activation. The first reported second-generation CARs featured CD28 signaling domains and the CD28 transmembrane sequence. This motif was used in third-generation CARs containing CD137 (4-1BB) signaling motifs as well (Zhao Y et al J Immunol. 2009; 183 (9): 5563-74). With the advent of new technology, the activation of T cells with beads linked to anti-CD3 and anti-CD28 antibody, and the presence of the canonical “signal 2” from CD28 was no longer required to be encoded by the CAR itself. Using bead activation, third-generation vectors were found to be not superior to second-generation vectors in in vitro assays, and they provided no clear benefit over second-generation vectors in mouse models of leukemia (Haso W, Lee D W, Shah N N, Stetler-Stevenson M, Yuan C M, Pastan I H, Dimitrov D S, Morgan R A, FitzGerald D J, Barrett D M, Wayne A S, Mackall C L, Orentas R J. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia, Blood. 2013; 121 (7):1165-74; Kochenderfer J N et al. Blood. 2012; 119 (12):2709-20). This is borne out by the clinical success of CD19-specific CARs that are in a second generation CD28/CD3-ζ (Lee D W et al. American Society of Hematology Annual Meeting. New Orleans, La.; Dec. 7-10, 2013) and CD137/CD3-ζ signaling formats (Porter D L et al. N Engl J Med. 2011; 365 (8): 725-33). In addition to CD137, other tumor necrosis factor receptor superfamily members such as OX40 also are able to provide important persistence signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009; 15(18):5852-60). Equally important are the culture conditions under which the CAR T-cell populations were cultured, for example the inclusion of the cytokines IL-2, IL-7, and/or IL-15 (Kaiser A D et al. Cancer Gene Ther. 2015; 22(2):72-78.
Current challenges in the more widespread and effective adaptation of CAR therapy for cancer relate to a paucity of compelling targets. Creating binders to cell surface antigens is now readily achievable, but discovering a cell surface antigen that is specific for tumor while sparing normal tissues remains a formidable challenge. One potential way to imbue greater target cell specificity to CAR-expressing T cells is to use combinatorial CAR approaches. In one system, the CD3-ζ and CD28 signal units are split between two different CAR constructs expressed in the same cell; in another, two CARs are expressed in the same T cell, but one has a lower affinity and thus requires the alternate CAR to be engaged first for full activity of the second (Lanitis E et al. Cancer Immunol Res. 2013; 1(1):43-53; Kloss C C et al. Nat Biotechnol. 2013; 31(1):71-5). A second challenge for the generation of a single ScFv-based CAR as an immunotherapeutic agent is tumor cell heterogeneity. At least one group has developed a CAR strategy for glioblastoma whereby the effector cell population targets multiple antigens (HER2, IL-13Ra, EphA2) at the same time in the hope of avoiding the outgrowth of target antigen-negative populations. (Hegde M et al. Mol Ther. 2013; 21(11):2087-101).
T-cell-based immunotherapy has become a new frontier in synthetic biology; multiple promoters and gene products are envisioned to steer these highly potent cells to the tumor microenvironment, where T cells can both evade negative regulatory signals and mediate effective tumor killing. The elimination of unwanted T cells through the drug-induced dimerization of inducible caspase 9 constructs with chemical-based dimerizers, such as AP1903, demonstrates one way in which a powerful switch that can control T-cell populations can be initiated pharmacologically (Di Stasi A et al. N Engl J Med. 2011; 365(18):1673-83). The creation of effector T-cell populations that are immune to the negative regulatory effects of transforming growth factor-β by the expression of a decoy receptor further demonstrates the degree to which effector T cells can be engineered for optimal antitumor activity (Foster A E et al. J Immunother. 2008; 31(5):500-5). Thus, while it appears that CARs can trigger T-cell activation in a manner similar to an endogenous T-cell receptor, a major impediment to the clinical application of this technology to date has been limited in vivo expansion of CAR+ T cells, rapid disappearance of the cells after infusion, and disappointing clinical activity. This may be due in part to the murine origin of some of the CAR sequences employed.
The requirement of patients who have received either antibody or CAR-T therapy to subsequently undergo HSCT in order to maintain durable responses remains an area of active debate. Although high responses are reported for CD19 CAR-T trials, at least 20% of patients fail in the near-term (Davis K L, Mackall C L, 2016, Blood Advances 1:265-268). The best results at 12 months post-CAR19 treatment reported show a RFS of 55% and OS of 79% in patients who were able to receive the T cell product at the University of Pennsylvania (Maude S L, Teachey D T, Rheingold S R, Shaw P A, Aplenc R, Barrett D M, Barker C S, Callahan C, Frey N V, Farzana N, Lacey S F, Zheng A, Levine B, Melenhorst J J, Motley L, Prter D L, June C H, Grupp S A, 2016, J Clin Oncol 34, no.15_suppl (May 2016) 3011-3011). Given the expected long term responses of 50% or less, there remains significant clinical need for new B cell malignancy targets such as ROR1.
The present invention addresses these needs by providing CAR compositions and therapeutic methods that can be used to treat cancers and other diseases and/or conditions. In particular, the present invention as disclosed and described herein provides CARs that may be used for the treatment of diseases, disorders or conditions associated with dysregulated expression of ROR1 and which CARs contain ROR1 antigen binding domains that exhibit a high surface expression on transduced T cells, exhibit a high degree of cytolysis of ROR1-expressing cells, and in which the transduced T cells demonstrate in vivo expansion and persistence.